Calcium-tolerant rabbit cardiomyocytes were isolated using retrograde aortic perfusion with a nominally
calcium-free,
collagenase buffer. In vitro ischemic preconditioning was induced by a 10-min episode of ischemic pelleting, followed by a 15-min post-incubation and a prolonged period of ischemic pelleting. Injury was assessed by determination of cell
contracture and
trypan blue permeability following hypotonic swelling and correlated with metabolic assays of
lactate and
adenine nucleotides. The
protein phosphatase PP1/2A inhibitor
calyculin A and PP2A-selective
fostriecin protected isolated rabbit cardiomyocytes from lethal injury after a 10-min pre-incubation and when added late into ischemic pellets after a delay of 75 min. At the time of late
drug addition, cells were severely
ATP-depleted and in rigor
contracture. Protection with
Calyculin A from 1 nM to 1 microM was dose-related. Cells pre-incubated with 10 nM to 10 microM
fostriecin 10 min prior to ischemic pelleting were protected with an EC50 approximating 71 nM, implying protection at a PP2A-selective dose. The selective
protein kinase C inhibitor,
calphostin C, blocked ischemic preconditioning protection but not protection from 1 microM
calyculin A. Protection of severely ischemic cardiomyocytes following
protein phosphatase inhibition appears not to require PKC activity or
ATP conservation. Pre-incubation of cells with
calyculin A induced high levels of phosphorylation in
p38 mitogen activated protein kinase (MAPK), as compared to the
ischemia-induced phosphorylation observed in the untreated group only at 30 min of
ischemia, providing evidence of
protein phosphatase activity in cardiomyocytes. Pharmacological protection in late
ischemia has been demonstrated, but the mechanism of protection is undetermined.